Computational protocol: A method for accurate detection of genomic microdeletions using real-time quantitative PCR

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Protocol publication

[…] Primers (Table ) were designed using Primer Express v2.0 (Applied Biosystems). The parameters for primer design were as follows; amplicons of 100–250 bp with a penalty score no higher than 10–12; primer melting temperature (Tm) range 58°C to 60°C; primer length range 18 to 25 bp, optimal 20 bp; primer G/C content range 20% to 60%; amplicon maximum Tm of 85°C. Runs of more than three identical nucleotides were avoided as polyG or polyC stretches can promote non-specific annealing, whilst runs of polyA and polyT can potentially open up stretches of the primer-template complex. Primers were required to be free of self-complementary sequence in order to avoid hairpin loop formation. In addition, no more than two G and/or C bases were permitted in the last five nucleotides at the 3' end to avoid GC clamp formation. Minimal deviation from the parameters was allowed only when there were no other options due to the complex nature of the 22q11.2 sequence. Primer and amplicon sequences were compared to the human genome using the BLAT program to guarantee that they showed 100% homology to only the sequence from which they were designed and also to guarantee that the forward and reverse primers were free of single nucleotide polymorphisms. […]

Pipeline specifications

Software tools Primer Express, BLAT
Applications Amplicon sequencing analysis, qPCR
Organisms Homo sapiens
Diseases Genetic Diseases, Inborn